The present invention relates to the field of HIV diagnosis. More particularly, the present invention relates to the field of diagnosing the susceptibility of an HIV sample to antiviral drugs used to treat HIV infection.
The present invention relates to a method for the rapid and reliable detection of drug-selected mutations in the HIV protease gene allowing the simultaneous characterization of a range of codons involved in drug resistance using specific sets of probes optimized to function together in a reverse-hybridization assay.
The human immunodeficiency virus (HIV) is the ethiological agent for the acquired immunodeficiency syndrome (AIDS). HIV, like other retroviruses, encodes an aspartic protease that mediates the maturation of the newly produced viral particle by cleaving viral polypeptides into their functional forms (Hunter et al). The HIV protease is a dimeric molecule consisting of two identical subunits each contributing a catalytic aspartic residue (Navia et al, Whodawer et al, Meek et al). Inhibition of this enzyme gives rise to noninfectious viral particles that cannot establish new cycles of viral replication (Kohl et al, Peng et al).
Attempts to develop inhibitors of HIV-1 protease were initially based on designing peptide compounds that mimicked the natural substrate. The availability of the 3-dimensional structure of the enzyme have more recently allowed the rational design of protease inhibitors (PI) using computer modeling (Huff et al, Whodawer et al). A number of second generation PI that are partially peptidic or entirely nonpeptidic have proven to exhibit particularly potent antiviral effects in cell culture. Combinations of various protease inhibitors with nucleoside and non-nucleoside RT inhibitors have also been studied extensively in vitro. In every instance, the combinations have been at least additive and usually synergistic.
In spite of the antiviral potency of many recently developed HIV-1 PI, the emergence of virus variants with decreased sensitivity to these compounds has been described both in cell culture and in treated patients thereby escaping the inhibitory effect of the antiviral (Condra et al.). Emergence of resistant variants depends on the selective pressure applied to the viral population. In the case of a relatively ineffective drug, selective pressure is low because replication of both wild-type virus and any variants can continue. If a more effective drug suppresses replication of virus except for a resistant variant, then that variant will be selected. Virus variants that arise from selection by PI carry several distinct mutations in the protease coding sequence that appear to emerge sequentially. A number of these cluster near the active site of the enzyme while others are found at distant sites. This suggests conformational adaptation to primary changes in the active site and in this respect certain mutations that increase resistance to PI also decrease protease activity and virus replication.
Amongst the PI, the antiviral activity of the PI ritonavir (A-75925; ABT-538). nelfinavir (AG-1343), indinavir (MK-639; L735; L524) and saquinavir (Ro 31-8959) have been approved by the Food and Drug Administration and are currently under evaluation in clinical trials involving HIV-infected patients. The VX-487 (141W94) antiviral compound is not yet approved. The most important mutations selected for the above compounds and leading to gradually increasing resistance are found at amino acid (aa) positions 30 (D to N), 46 (M to I), 48 (G to V), 50 (I to V), 54 (I to A, I to V), 82 (V to A, or F, or I, or T), 84 (I to V) and 90 (L to M). Other mutations associated with drug resistance to the mentioned compounds have been described (Schinazi et al). Saquinavir-resistant variants, which usually carry mutations at amino acid positions 90 and/or 48, emerge in approximately 45% of patients after 1 year of monotherapy. Resistance appears to develop less frequently with higher doses of saquinavir. Resistance to indinavir and ritonavir requires multiple mutations; usually at greater than 3 and up to 11 sites, with more amino acid substitutions conferring higher levels of resistance. Resistant isolates usually carry mutations at codons 82, 84, or 90. In the case of ritonavir, the mutation at codon 82 appears fist in most patients. Although mutant virions resistant to saquinavir are not cross-resistant to indinavir or ritonavir, isolates resistant to indinavir are generally ritonavir resistant and visa versa. Resistance to either indinavir or ritonavir usually results in cross-resistance to saquinavir. Approximately one third of indinavir resistant isolates are cross-resistant to nelfinavir as well.
The regime for an efficient antiviral treatment is currently not clear at all. Patterns of reduced susceptibility to HIV protease inhibitors have been investigated in vitro by cultivating virus in the presence of PI. These data, however, do not completely predict the pattern of amino-acid changes actually seen in patients receiving PI. Knowledge of the resistance and cross-resistance patterns should facilitate selection of optimal drug combinations and selection of sequences with non-overlapping resistance patterns. This would delay the emergence of cross-resistant viral strains and prolong the duration of effective antiretroviral activity in patients. Therefore, there is need for methods and systems that detect these mutational events in order to give a better insight into the mechanisms of HIV resistance. Further, there is need for methods and systems which can provide data important for the antiviral therapy to follow in a more time-efficient and economical manner compared to the conventional cell-culture selection techniques.
It is an aim of the present invention to develop a rapid and reliable detection method for determination of the antiviral drug resistance of viruses, which contain protease genes such as HIV retroviruses present in a biological sample.
More particularly it is an aim of the present invention to provide a genotyping assay allowing the detection of the different HIV protease gene wild type and mutation codons involved in the antiviral resistance in one single experiment.
It is also an aim of the present invention to provide an HIV protease genotyping assay or method which allows to infer the nucleotide sequence at codons of interest and/or the amino acids at the codons of interest and/or the antiviral drug selected spectrum, and possibly also infer the HIV type or subtype isolate involved.
Even more particularly it is an aim of the present invention to provide a genotyping assay allowing the detection of the different HIV protease gene polymorphisms representing wild-type and mutation codons in one single experimental setup.
It is another aim of the present invention to select particular probes able to discriminate wild-type HIV protease sequences from mutated or polymorphic HIV protease sequences conferring resistance to one or more antiviral drugs, such as ritonavir (A-75925; ABT-538), nelfinavir (AG-1343), indinavir (MK-639; L735; L524), saquinavir (Ro 31-8959) and VX-478 (141W94) or others (Shinazi et al).
It is more particularly an aim of the present invention to select particular probes able to discriminate wild-type HIV protease sequences from mutated or polymorphic HIV protease sequences conferring resistance to ritonavir (A-75925; ABT-538).
It is more particularly an aim of the present invention to select particular probes able to discriminate wild-type HIV protease sequences from mutated HIV protease sequences conferring resistance to nelfinavir (AG-1343).
It is more particularly an aim of the present invention to select particular probes able to discriminate wild-type HIV protease sequences from mutated HIV protease sequences conferring resistance to indinavir (MK-639; L735; L524).
It is more particularly an aim of the present invention to select particular probes able to discriminate wild-type HIV protease sequences from mutated HIV protease sequences conferring resistance to saquinavir (Ro 31-8959).
It is more particularly an aim of the present invention to select particular probes able to discriminate wild-type HIV protease sequences from mutated HIV protease sequences conferring resistance to VX-478 (141W94).
It is also an aim of the present invention to select particular probes able to determine and/or infer cross-resistance to HIV protease inhibitors.
It is more particularly an aim of the present invention to select particular probes able to discriminate wild-type HIV protease from mutated HIV protease sequences involving at least one of amino acid positions 30 (D to N), 46 (M to I), 48 (G to V), 50 (I to V), 54 (I to A or V), 82 (V to A or F or I or T), 84(I to V) and 90 (L to M) of the viral protease gene.
It is particularly an aim of the present invention to select a particular set of probes, able to discriminate wild-type HIV protease sequences from mutated HIV protease sequences conferring resistance to any of the antiviral drugs defined above with this particular set of probes being used in a reverse hybridization assay.
It is moreover an aim of the present invention to combine a set of selected probes able to discriminate wild-type HIV protease sequences from mutated HIV protease sequences conferring resistance to antiviral drugs with another set of selected probes able to identify the HIV isolate, type or subtype present in the biological sample, whereby all probes can be used under the same hybridization and wash-conditions.
It is also an aim of the present invention to select primers enabling the amplification of the gene fragment(s) determining the antiviral drug resistance trait of interest.
It is also an aim of the present invention to select particular probes able to identify mutated HIV protease sequences resulting in cross-resistance to antiviral drugs.
The preset invention also aims at diagnostic kits comprising said probes useful for developing such a genotyping assay.
The present invention also aims at diagnostic kits comprising said primers useful for developing such a genotyping assay.
All the aims of the present invention have been met by the following specific embodiments.
According to one embodiment, the present invention relates to a method for determining the susceptibility to antiviral drugs of HIV viruses in a biological sample, with said method comprising:
a) if need be, releasing, isolating or concentrating the polynucleic acids present in the sample;
b) if need be amplifying the relevant part of the protease gene of HIV with at least one suitable primer pair;
c) hybridizing the polynucleic acids of step a) or b) with at least one of the following probes:
probes specifically hybridizing to a target sequence comprising codon 30;
probes specifically hybridizing to a target sequence comprising codon 46 and/or 48;
probes specifically hybridizing to a target sequence comprising codon 50;
probes specifically hybridizing to a target sequence comprising codon 54;
probes specifically hybridizing to a target sequence comprising codon 82 and/or 84;
probes specifically hybridizing to a target sequence comprising codon 90;
or the complement of said probes,
further characterized in that said probes specifically hybridize to any of the target sequences presented in FIG. 1, or to the complement of said target sequences;
d) inferring from the result of step c) whether or not a mutation giving rise to drug resistance is present in any of said target sequences.
The numbering of HIV-1 protease gene encoded amino acids is as generally accepted in literature. Mutations that give rise to an amino acid change at position 48 or 90 are known to confer resistance to saquinavir (Eriebe et al; Tisdale et al). An amino acid change at codon 46 or 54 or 82 or 84 results in ritonavir and indinavir resistance (Kempf et al; Emini et al; Condra et al). Amino acid changes at positions 30 and 46 confer resistance to nelfinavir (Patick et al) and amino acid changes at position 50 confers resistance to VX-487 (Rao et al). Therefore, the method described above allows to determine whether a HIV strain is susceptible or resistant to any of the drugs mentioned above. This method can be used, for instance, to screen for mutations conferring resistance to any of the mentioned drugs before initiating therapy. This method may also be used to s for mutations that may arise during the course of therapy (i.e. monitoring of drug therapy). It is obvious that this method may also be used to determine resistance to drugs other than the above-mentioned drugs, provided that resistance to these other drugs is linked to mutations that can be detected by use of this method. This method may also be used for the specific detection of polymorphic nucleotides. It is to be understood that the said probes may only partly overlap with the targets sequences of FIG. 1, table 2 and table 3, as long as they allow for specific detection of the relevant polymorphic nucleotides as indicated above. The sequences of FIG. 1, table 2 and table 3 were derived from polynucleic acid fragments comprising the protease gene. These fragments were obtained by PCR amplification and were inserted into a cloning vector and sequence analyzed as described in example 1. It is to be noted that some polynucleic acid fragments comprised polymorphic nucleotides in their sequences, which have not been previously disclosed. These novel polymorphic nucleotide sequences are represented in table 4 below.
The present invention thus also relates to these novel sequences, or a fragment thereof, wherein said fragment consists of at least 10, preferably 15, even more preferably 20 contiguous nucleotides and contains at least one polymorphic nucleotide. It is furthermore to be understood that these new polymorphic nucleotides may also be expected to arise in another sequence context than in the mentioned sequences. For instance a G at the third position of codon 55 is shown in SEQ ID No 478 m combination with a T at the third position of codon 54, but a G at the third position of codon 55 may also be expected to occur in the context of a wild type sequence. It is also to be understood that the above mentioned specifications apply to the complement of the said target sequences as well. This applies also to FIG. 1.
According to a preferred embodiment the present invention relates to a method as indicated above, further characterized in that said probes are capable of simultaneously hybridizing to their respective target regions under appropriate hybridization and wash conditions allowing the detection of the hybrids formed.
According to a preferred embodiment, step c is performed using a set of at least 2, preferably at least 3, more preferably at least 4 and most preferably at least 5 probes meticulously designed as such that they show the desired hybridization results. In general this method may be used for any purpose that relies on the presence or absence of mutations that can be detected by this method, e.g. for genotyping. The probes of table 1 have been optimized to give specific hybridization results when used in a LiPA assay (see below), as described in examples 2 and 3. These probes have thus also been optimized to simultaneously hybridize to their respective target regions under the same hybridization and wash conditions allowing the detection of hybrids. The sets of probes for each of the codons 30,46/48, 50, 54 and 82/84 have been tested experimentally as described in examples 2 and 3. The reactivity of the sets shown in table 1 with 856 serum samples from various geographic origins was evaluated. It was found that the sets of probes for codons 30, 46/48, 50, 54 and 82/84 reacted with 98.9%, 99.6%, 98.5%, 99.20%, 95.4% and 97.2% of the test samples, respectively. The present invention thus also relates to the sets of probes for codons 30, 46/48, 50, 54, 82/84 and 90, shown in table 1 and table 7.
According to another even more preferred embodiment, the present invention relates to a method as defined above, further characterized in that:
step b) comprises amplifying a fragment of the protease gene with at least one 5xe2x80x2-primer specifically hybridizing to a target sequence located between nucleotide position 210 and nucleotide position 260 (codon 87), more preferably between nucleotide position 220 and nucleotide position 260 (codon 87), more preferably between nucleotide position 230 and nucleotide position 260 (codon 87), even more preferably at nucleotide position 241 to nucleotide position 260 (codon 87) in combination with at least one suitable 3xe2x80x2-primer, and
step c) comprises hybridizing the polynucleic acids of step a) or b) with at least one of the probes specifically hybridizing to a target sequence comprising codon 90.
According to another even more preferred embodiment, the present invention relates to a method as defined above, further characterized in that:
step b) comprises amplifying a fragment of the protease gene with at least one 3xe2x80x2-primer specifically hybridizing to a target sequence located between nucleotide position 253 (codon 85) and nucleotide positions 300, more preferably between nucleotide position 253 (codon 85) and nucleotide positions 290, more preferably between nucleotide position 253 (codon 85) and nucleotide positions 280, even more preferably at nucleotide position 253 (codon 85) to nucleotide position 273 (codon 91), in combination with at least one suitable 5xe2x80x2-primer, and
step c) comprises hybridizing the polynucleic acids of step a) or b) with at least one of the probes specifically hybridizing to a target sequence comprising any of codons 30, 46, 48, 50, 52, 54, 82 and 84.
It has been found, unexpectedly, that an amplified nucleic acid fragment comprising all of the above-mentioned codons, does not hybridize optimally to probes comprising codon 82, 84 or 90. On the other hand, a shorter fragment, for instance the fragment which is amplified by use of the primers Prot41bio and Prot6bio with respectively seq id no 5 and seq id no 4; hybridizes better to probes comprising codon 90. Better hybridization is also obtained when the fragment is amplified with primer Prot41bio in combination with primers Prot6abio, Prot6bbio, Prot6cbio and Prot6dbio. The present invention thus also relates to a method as defined above, finder characterized in that the 5xe2x80x2-primer is seq id no 5 and at least one 3xe2x80x2 primer is chosen from seq id no 4, seq id no506, seq id no 507, seq id no 508, and seq id 509. Likewise, another shorter fragment, for instance the fragment which is amplified by use of the primers Prot2bio and Prot31bio with respectively seq id no 3 and seq id no 6, was found to hybridize better to probes comprising codon 82 and/or 84. Hence the present invention also relates to a method as defined above, further characterized in that the 5xe2x80x2-primer is seq id no 5 and at least one 3xe2x80x2-primer is chosen from seq id no 4, seq id no506, seq id no 507, seq id no 508, and seq id no 509.
New sets of amplification primers as mentioned in example 1 were selected. The present invention thus also relates to primers: prot16 (SEQ ID NO 501), prot5 (SEQ ID NO 5), prot2abio (SEQ ID NO 503), prot2bbio (SEQ ID NO 504), prot31bio (SEQ ID NO 6), prot41-bio (SEQ ID NO 505), prot6a (SEQ ID NO 506), prot6b (SEQ ID NO 507), prot6c (SEQ ID NO 508) and prot6d (SEQ DID NO 509). A number of these primers are chemically modified (biotinylated), others are not. The present invention relates to any of the primers mentioned, primers containing unmodified nucleotides, or primers containing modified nucleotides.
Different techniques can be applied to perform the sequence-specific hybridization methods of the present invention. These techniques may comprise immobilizing the amplified HIV polynucleic acids on a solid support and performing hybridization with labeled oligonucleotide probes. HIV polynucleic acids may also be immobilized on a solid support without prior amplification and subjected to hybridization. Alternatively, the probes may be immobilized on a solid support and hybridization may be performed with labeled HIV polynucleic acids, preferably after amplification. This technique is called reverse hybridization. A convenient reverse hybridization technique is the line probe assay (LiPA). This assay uses oligonucleotide probes immobilized as parallel lines on a solid support strip (Stuyver et al., 1993). It is to be understood that any other technique based on the above-mentioned methods is also covered by the present invention.
According to another preferred embodiment, the present invention relates to any of the probes mentioned above and/or to any of the primers mentioned above, with said primers and probes being designed for use in a method for determining the susceptibility to antiviral drugs of HIV viruses in a sample. According to an even more preferred embodiment, the present invention relates to the probes with seq id no 7 to seq id no 477 and seq id no510 to seq id no 519, more preferably to the seq id no mentioned in Table 1 and Table 7, and to the primers with seq id no 3, 4, 5 and 6, 501, 502, 503, 504, 505, 506, 507, 508 and 509. The skilled man will recognize that addition or deletion of one or more nucleotides at their extremities may adapt the said probes and primers. Such adaptations may be requited if the conditions of amplification or hybridization are changed, or if the amplified material is RNA instead of DNA, as is the case in the NASBA system
According to another preferred embodiment, the present invention relates to a diagnostic kit enabling a method for determining the susceptibility to antiviral drugs of HIV viruses in a biological sample, with said kit comprising:
a) when appropriate, a means for releasing, isolating or concentrating the polynucleic acids present in said sample;
b) when appropriate, at least one of the primers of any of claims 4 to 6;
c) at least one of the probes of any of claims 1 to 3, possibly fixed to a solid support;
d) a hybridization buffer, or components necessary for producing said buffer;
e) a wash solution, or components necessary for producing said solution;
f) when appropriate, a means for detecting the hybrids resulting from the preceding hybridization;
h) when appropriate, a means for attaching said probe to a solid support.
The following definitions serve to illustrate the terms and expressions used in the present invention.
The term xe2x80x9cantiviral drugsxe2x80x9d refers particularly to any antiviral protease inhibitor. Examples of such antiviral drugs and the mutation they may cause in the HIV protease gene are disclosed in Schinazi et al., 1997. The contents of the latter two documents particularly are to be considered as forming part of the present invention. The most important antiviral drugs focussed at in the present invention are disclosed in Tables 1 to 2.
The target material in the samples to be analyzed may either be DNA or RNA, e.g.: genomic DNA, messenger RNA, viral RNA or amplified versions thereof. These molecules are also termed polynucleic acids.
It is possible to use genomic DNA or RNA molecules from HIV samples in the methods according to the present invention.
Well-known extraction and purification procedures are available for the isolation of RNA or DNA from a sample (fi. in Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbour Laboratory Press (1989)).
The term xe2x80x9cprobexe2x80x9d refers to single stranded sequence-specific oligonucleotides, which have a sequence, which is complementary to the target sequence to be detected.
The term xe2x80x9ctarget sequencexe2x80x9d as referred to in the present invention describes the wild type nucleotide sequence, or the sequence comprising one or more polymorphic nucleotides of the protease gene to be specifically detected by a probe according to the present invention. This nucleotide sequence may encompass one or several nucleotide changes. Target sequences may refer to single nucleotide positions, codon positions, nucleotides encoding amino acids or to sequences spanning any of the foregoing nucleotide positions. In the present invention said target sequence often includes one or two variable nucleotide positions.
The term xe2x80x9cpolymorphic nucleotidexe2x80x9d indicates a nucleotide in the protease gene of a particular HIV virus that is different from the nucleotide at the corresponding position in at least one other HIV virus. The polymorphic nucleotide may or may not give rise to resistance to an antiviral drug. It is to be understood that the complement of said target sequence is also a suitable target sequence in some cases. The target sequences as defined in the present invention provide sequences which should be complementary to the central part of the probe which is designed to hybridize specifically to said target region.
The term xe2x80x9ccomplementaryxe2x80x9d as used herein means that the sequence of the single stranded probe is exactly the (inverse) complement of the sequence of the single-stranded target, with the target being defined as the sequence where the mutation to be detected is located.
xe2x80x9cSpecific hybridizationxe2x80x9d of a probe to a target sequence of the HIV polynucleic acids means that said probe forms a duplex with part of this region or with the entire region under the experimental conditions used, and that under those conditions said probe does not form a duplex with other regions of the polynucleic acids present in the sample to be analyzed.
Since the current application requires the detection of single basepair mismatches, very stringent conditions for hybridization are required, allowing in principle only hybridization of exactly complementary sequences. However, variations arm possible in the length of the probes (see below), and it should be noted that, since the central part of the probe is essential for its hybridization characteristics, possible deviations of the probe sequence versus the target sequence may be allowable towards head and tail of the probe, when longer probe sequences are used. These variations, which may be conceived from the common knowledge in the art, should however always be evaluated experimentally, in order to check if they result in equivalent hybridization characteristics than the exactly complementary probes.
Preferably, the probes of the invention are about 5 to 50 nucleotides long, more preferably from about 10 to 25 nucleotides. Particularly preferred lengths of probes include 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. The nucleotides as used in the present invention may be ribonucleotides, deoxyribonucleotides and modified nucleotides such as inosine or nucleotides containing modified groups, which do not essentially alter their hybridization characteristics.
Probe sequences are represented throughout the specification as single stranded DNA oligonucleotides from the 5xe2x80x2 to the 3xe2x80x2 end. It is obvious to the man skilled in the art that any of the below-specified probes can be used as such, or in their complementary form, or in their RNA form (wherein U replaces T).
The probes according to the invention can be prepared by cloning of recombinant plasmids containing inserts including the corresponding nucleotide sequences, if need be by cleaving the latter out from the cloned plasmids upon using the adequate nucleases and recovering them, e.g. by fractionation according to molecular weight. The probes according to the present invention can also be synthesized chemically, for instance by the conventional phospho-triester method.
The term xe2x80x9csolid supportxe2x80x9d can refer to any substrate to which an oligonucleotide probe can be coupled, provided that it retains its hybridization characteristics and provided that the background level of hybridization remains low. Usually the solid substrate will be a microtiter plate, a membrane (e.g. nylon or nitrocellulose) or a microsphere (bead) or a chip. Prior to application to the membrane or fixation it may be convenient to modify the nucleic acid probe in order to facilitate fixation or improve the hybridization efficiency. Such modifications may encompass homopolymer tailing, coupling with different reactive groups such as aliphatic groups, NH2 groups, SH groups, carboxylic groups, or coupling with biotin, haptens or proteins.
The term xe2x80x9clabeledxe2x80x9d refers to the use of labeled nucleic acids. Labeling may be carried out by the use of labeled nucleotides incorporated during the polymerase step of the amplification such as illustrated by Saiki et al. (1988) or Bej et al. (1990) or labeled primers, or by any other method known to the person skilled in the art. The nature of the label may be isotopic (32P, 35S, etc.) or non-isotopic (biotin, digoxigenin, etc.).
The term xe2x80x9cprimerxe2x80x9d refers to a single stranded oligonucleotide sequence capable of acting as a point of initiation for synthesis of a primer extension product, which is complementary to the nucleic acid strand to be copied. The length and the sequence of the primer must be such that they allow to prime the synthesis of the extension products. Preferably the primer is about 5-50 nucleotides long. Specific length and sequence will depend on the complexity of the required DNA or RNA targets, as well as on the conditions of primer use such as temperature and ionic strength.
The term xe2x80x9cprimer pairxe2x80x9d refers to a set of primers comprising at least one 5xe2x80x2 primer and one 3xe2x80x2 primer. The primer pair may consist of more than two primers, the complexity of the number of primers will depend on the hybridization conditions, variability of the sequences in the regions to be amplified and the target sequences to be detected.
The fact that amplification primers do not have to match exactly with the corresponding template sequence to warrant proper amplification is amply documented in the literature (Kwok et al., 1990).
The amplification method used can be either polymerase chain reaction (PCR; Saiki et al., 1988), ligase chain reaction (LCR; Landgren et al., 1988; Wu and Wallace, 1989; Barany, 1991), nucleic acid sequence-based amplification (NASBA; Guatelli et al., 1990; Compton, 1991), transcription-based amplification system (TAS; Kwoh et al., 1989), strand displacement amplification (SDA; Duck, 1990) or amplification by means of QB replicase (Lomeli et al., 1989) or any other suitable method to amplify nucleic acid molecules known in the art.
The oligonucleotides used as primers or probes may also comprise nucleotide analogues such as phosphorothiates (Matsukura et al., 1987), alkylphosphorothiates (Miller et al., 1979) or peptide nucleic acids (Nielsen et al., 1991; Nielsen et al., 1993) or may contain intercalating agents (Asseline et al., 1984).
As most other variations or modifications introduced into the original DNA sequences of the invention these variations will necessitate adaptations with respect to the conditions under which the oligonucleotide should be used to obtain the required specificity and sensitivity. However the eventual results of hybridization will be essentially the same as those obtained with the unmodified oligonucleotides.
The introduction of these modifications may be advantageous in order to positively influence characteristics such as hybridization kinetics, reversibility of the hybrid-formation, biological stability of the oligonucleotide molecules, etc.
The xe2x80x9csamplexe2x80x9d may be any biological material taken either directly from the infected human being (or animal), or after culturing (enrichment). Biological material may be e.g. expectorations of any kind, broncheolavages, blood, skin tissue, biopsies, sperm, lymphocyte blood culture material, colonies, liquid cultures, fecal samples, urine etc.
The sets of probes of the present invention will include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more probes. Said probes may be applied in two or more distinct and known positions an a solid substrate. Often it is preferable to apply two or more probes together in one and the same position of said solid support.
For designing probes with desired characteristics, the following useful guidelines known to the person skilled in the art can be applied.
Because the extent and specificity of hybridization reactions such as those described herein are affected by a number of factors, manipulation of one or more of those factors will determine the exact sensitivity and specificity of a particular probe, whether perfectly complementary to its target or not. The importance and effect of various assay conditions, explained further herein, are known to those skilled in the art.
The stability of the [probe:target] nucleic acid hybrid should be chosen to be compatible with the assay conditions. This may be accomplished by avoiding long AT-rich sequences, by terminating the hybrids with G:C base pairs, and by designing the probe with an appropriate Tm. The beginning and end points of the probe should be chosen so that the length and % GC result in a Tm about 2-10xc2x0 C. higher than the temperature at which the final assay will be performed. The base composition of the probe is significant because G-C base pairs exhibit greater thermal stability as compared to A-T base pairs due to additional hydrogen bonding. Thus, hybridization involving complementary nucleic acids of higher G-C content will be stable at higher temperatures.
Conditions such as ionic strength and incubation temperature under which a probe will be used should also be taken into account when designing a probe. It is known that hybridization will increase as the ionic strength of the reaction mixture increases, and that the thermal stability of the hybrids will increase with increasing ionic strength. On the other hand, chemical reagents, such as formamide, urea, DMSO and alcohols, which disrupt hydrogen bonds, will increase the stringency of hybridization. Destabilization of the hydrogen bonds by such reagents can greatly reduce the Tm. In general, optimal hybridization for synthetic oligonucleotide probes of about 10-50 bases in length occurs approximately 5xc2x0 C. below the melting temperature for a given duplex. Incubation at tempts below the optimum may allow mismatched base sequences to hybridize and can therefore result in reduced specificity.
It is desirable to have probes, which hybridize only under conditions of high stringency. Under high stringency conditions only highly complementary nucleic acid hybrids will form; hybrids without a sufficient degree of complementarity will not form. Accordingly, the stringency of the assay conditions determines the amount of complementarity needed between two nucleic acid strands forming a hybrid. The degree of stringency is chosen such as to maximize the difference in stability between the hybrid formed with the target and the nontarget nucleic acid. In the present case, single base pair changes need to be detected, which requires conditions of very high stringency.
The length of the target nucleic acid sequence and, accordingly, the length of the probe sequence can also be important. In some cases, there may be several sequences from a particular region, varying in location and length, which will yield probes with the desired hybridization characteristics. In other cases, one sequence may be significantly better than another that differs merely by a single base. While it is possible for nucleic acids that are not perfectly complementary to hybridize, the longest stretch of perfectly complementary base sequence will normally primarily determine hybrid stability. While oligonucleotide probes of different lengths and base composition may be used, preferred oligonucleotide probes of this invention arm between about 5 to 50 (more particularly 10-25) bases in length and have a sufficient stretch in the sequence which is perfectly complementary to the target nucleic acid sequence.
Regions in the target DNA or RNA, which are known to form strong internal structures inhibitory to hybridization, arc less preferred. Likewise, probes with extensive self-complementarity should be avoided. As explained above, hybridization is the association of two single strands of complementary nucleic acids to form a hydrogen bonded double strand. It is implicit that if one of the two strands is wholly or partially involved in a hybrid that it will be less able to participate in formation of a new hybrid. There can be intramolecular and intermolecular hybrids formed within the molecules of one type of probe if there is sufficient self complementarity. Such structures can be avoided through careful probe design. By designing a probe so that a substantial portion of the sequence of interest is single stranded, the rate and extent of hybridization may be greatly increased. Computer programs are available to search for this type of interaction. However, in certain instances, it may not be possible to avoid this type of interaction.
Standard hybridization and wash conditions are disclosed in the Materials and Methods section of the Examples. Other conditions are for instance 3xc3x97SSC (Sodium Saline Citrate), 20% deionized FA (Formamide) at 50xc2x0 C.
Other solutions (SSPE (Sodium saline phosphate EDTA), TMACl (tetramethyl ammonium Chloride), etc.) and temperatures can also be used provided that the specificity and sensitivity of the probes is maintained. If need be, slight modifications of the probes in length or in sequence have to be carried out to maintain the specificity and sensitivity required under the given circumstances.
Primers may be labeled with a label of choice (e.g. biotin). Different primer-based target amplification systems may be used, and preferably PCR-amplification, as set out in the examples. Single-round or nested PCR may be used.
The term xe2x80x9chybridization bufferxe2x80x9d means a buffer enabling a hybridization reaction to occur between the probes and the polynucleic acids present in the sample, or the amplified product, under the appropriate stringency conditions.
The term xe2x80x9cwash solutionxe2x80x9d means a solution enabling washing of the hybrids formed under the appropriate stringency conditions.
The following examples only serve to illustrate the present invention. These examples are in no way intended to limit the scope of the present invention.